We are continuing our study of the E. coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: these include T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41, 61, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and accessory proteins on the primer template. Using a system in which a photoactivatable aryl azide can be moved stepwise through a thirty base region of the primer, we find that the accessory proteins and 32 protein can be cross-linked to the primer in the presence of a non-hydrolyzable ATP analogue. The initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of each of the accessory proteins to yield a complex capable of processive DNA synthesis. Protein-protein interactions required for strand displacement synthesis. Mutations in the T4 DNA polymerase can decrease its ability to interact with the proteins that allow strand displacement synthesis with the wild type enzyme. The purified tsLl4l mutant polymerase (alanine changed to valine at 737) is stimulated by the accessory proteins and 32 protein, but is arrested at pause sites on single-stranded templates more frequently that the wild type, and carries out practically no strand displacement synthesis on a forked DNA template with the accessory proteins, 32 protein, and the 41 protein helicase. We have recently cloned and purified to homogeneity the T4 gene 59 protein, which increases DNA unwinding by the 41 helicase. We find that addition of this 59 protein is absolutely required for strand displacement synthesis with the tsL141 mutant polymerase. The position of the tsL141 mutation (737) and that of a second site mutation (771) that suppresses its phenotype are C-terminal to the regions where T4 polymerase shares homology with eukaryotic DNA polymerases, and may be in a region important for its interactions with the other T4 replication proteins.